Summary
This paper presents a method for determining the nucleotide sequence of DNA. The central thesis is that DNA polymerase can be primed to synthesize a complementary strand of DNA, and by using chain-terminating nucleotides, fragments of specific lengths corresponding to each nucleotide can be generated and then separated by size to reveal the sequence. The method involves using a primer, DNA polymerase, a DNA template, deoxynucleotide triphosphates (dNTPs), and a small amount of one dideoxynucleotide triphosphate (ddNTP). The inclusion of ddNTPs at specific positions halts strand elongation, creating a collection of DNA fragments ending at every instance of that particular nucleotide.
The key idea is the controlled termination of DNA synthesis. By performing this reaction four times, each with a different ddNTP (ddATP, ddTTP, ddCTP, ddGTP), four sets of fragments are produced, each set terminating at a specific base. These fragments are then separated by gel electrophoresis. The resulting pattern of bands allows for the direct reading of the DNA sequence from the gel. Readers understand a practical, efficient technique for DNA sequencing that was foundational to molecular biology.
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Key concepts
- Primed synthesis — DNA polymerase extends a pre-existing short nucleotide chain (primer) using a DNA template.
- Chain termination — The incorporation of a dideoxynucleotide (lacking a 3'-hydroxyl group) prevents further elongation of the DNA strand.
- DNA polymerase — An enzyme that synthesizes DNA molecules from nucleotides, building a new strand complementary to a template strand.
- Gel electrophoresis — A technique used to separate DNA fragments based on their size and charge.